TNFα protects cardiac mitochondria independently of its cell surface receptors

Our novel proposal is that TNF alpha exerts a direct effect on mitochondrial respiratory function in the heart, independently of its cell surface receptors. TNF alpha-induced cardioprotection is known to involve reactive oxygen species (ROS) and sphingolipids. We therefore further propose that this direct mitochondrial effect is mediated via ROS and sphingolipids. The protective concentration of TNF alpha (0.5 ng/ml) was added to isolated heart mitochondria from black 6 x 129 mice (WT) and double TNF receptor knockout mice (TNFR1&2(-/-)). Respiratory parameters and inner mitochondrial membrane potential were analyzed in the presence/absence of two antioxidants, N-acetyl-l-cysteine or N-tert-butyl-alpha-(2-sulfophenyl)nitrone or two antagonists of the sphingolipid pathway, N-oleoylethanolamine (NOE) or imipramine. In WT, TNF alpha reduced State 3 respiration from 279.3 +/- A 3 to 119.3 +/- A 2 (nmol O-2/mg protein/min), increased proton leak from 15.7 +/- A 0.6% (control) to 36.6 +/- A 4.4%, and decreased membrane potential by 20.5 +/- A 3.1% compared to control groups. In TNFR1&2(-/-) mice, TNF alpha reduced State 3 respiration from 205.2 +/- A 4 to 75.7 +/- A 1 (p < 0.05 vs. respective control). In WT mice, both antioxidants added with TNF alpha restored State 3 respiration to 269.2 +/- A 2 and 257.6 +/- A 2, respectively. Imipramine and NOE also restored State 3 respiration to 248.4 +/- A 2 and 249.0 +/- A 2, respectively (p < 0.01 vs. TNF alpha alone). Similarly, both antioxidant and inhibitors of the sphingolipid pathway restored the proton leak to pre-TNF values. TNF alpha-treated mitochondria or isolated cardiac muscle fibers showed an increase in respiration after anoxia-reoxygenation, but this effect was lost in the presence of an antioxidant or NOE. Similar data were obtained in TNFR1&2(-/-) mice. TNF alpha exerts a protective effect on respiratory function in isolated mitochondria subjected to an anoxia-reoxygenation insult. This effect appears to be independent of its cell surface receptors, but is likely to be mediated by ROS and sphingolipids.